Oxidative stress is a critical mediator of the injury response after neonatal hypoxia ischemia. Evidence suggests that hydrogen peroxide toxicity is related to cell death but a paradox exists such that H2O2 may also be a signaling molecule that is involved in neuroprotection. In particular, H2O2 may be a critical mediator of hypoxic or ischemic preconditioning in the immature brain. We hypothesize that H2O2 serves as a critical signaling molecule for activation of hypoxia inducible factor 1 (HIF1) to promote protection in the brain after neonatal ischemia. To address this hypothesis we will determine the mechanisms by which HIF1 is regulated by H2O2 to generate a cytoprotective response in immature primary neurons from hippocampus and cortex by measuring HIF 1a induction by mRNA and protein levels, DMA binding activity by EMSA and promoter activity. We will measure HIF1a dependent cytoprotective genes and compare to changes in pro- apoptotic genes and their protein targets. We will measure H2O2 and the cellular localization of reactive oxygen species and we will block preconditioning using antisense or knockdown strategies against HIF1. We will study redox-sensitive signaling pathways such as MARK, PI3K/Akt to determine their involvement in hypoxic HIF1 signaling and neuronal protection. We will assess phosphorylation states and use pharmacological inhibitors to determine relevant activities. The second aim will test how altering glutathione peroxidase activities by overexpression or knockdown will affect hypoxic preconditioning and injury in vitro and in vivo and how this relates to H1F1 activation and redox signaling. We will measure HIF1 activation and H2O2 levels necessary to achieve preconditioning neuroprotection. In the final aim we will determine how modulation of HIF1 activity by genetic deletion, pharmacological stabilization or induction by growth factors affect hypoxic preconditioning and neuronal injury in vivo. We will also test behavioral outcomes in these animals to determine whether this is a functional deficit. RELEVANCE TO PUBLIC HEALTH. Lack of oxygen to the newborn brain is the major cause of lifelong disability in children resulting in mental retardation, epilepsy and cerebral palsy. Understanding pathways for protecting the brain will result in therapeutic avenues for brain recovery.